The ability of V. cholerae to cause disease is dependent upon its ability to resist the antimicrobial compounds that are present in the host gastrointestinal tract in conjunction with the expression of essential virulence factors. We recently showed that the ability of V. cholerae to express virulence factors and resist antimicrobial compounds present in the host is dependent upon active efflux provided by RND family efflux systems. V. cholerae lacking its RND efflux systems was highly attenuated in the infant mouse model and produced reduced amounts of cholera toxin (CT) and the toxin coregulated pilus (TCP). Reduced CT and TCP production correlated to repression of the genes encoding the virulence regulators TcpPH and ToxT via a mechanism that was independent of quorum sensing. Collectively these results suggested that the RND efflux systems functioned in a novel virulence gene regulatory system. In this proposal we will characterize the role of the RND efflux systems in V. cholerae virulence gene expression and pathogenesis. In aim 1 we will identify genes that are involved in the RND-dependent repression of virulence factor production. In aim 2 we will identify the genes and environmental stimuli that affect the expression of the RND efflux systems. In aim 3 we will characterize the role of a novel molecule in the regulated expression of virulence factor production. These studies will define the role of the V. cholerae RND efflux systems in V. cholerae biology which will contribute to the development of new vaccines and cholera therapeutics.